The noradrenergic neurons of the locus coeruleus (LC) have been shown to play an important role in mediating physical opiate dependence and withdrawal. Over the last 7 years, the laboratories of Drs. Nestler, Aghajanian, Duman, and Alreja have carried out highly integrated biochemical and electrophysiological studies to investigate the molecular and cellular mechanisms underlying opiate tolerance, dependence, and withdrawal in these neurons. They have found that opiates acutely inhibit LC neurons via the regulation of two ion channel conductances, a K+ conductance and a non-specific cationic conductance. Opiate regulation of the latter conductance appears to be mediated via opiate-induced inhibition of the cAMP second messenger and protein phosphorylation pathway. In contrast to acute opiate action, chronic opiate administration has been shown to upregulate the cAMP pathway in the LC at every major step between receptor and physiological response, with increases observed in specific G-proteins, adenylate cyclase, cAMP-dependent protein kinase, and several substrates for the protein kinase including tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of catecholamines. Several lines of evidence now support the view that this upregulation of the cAMP pathway contributes to opiate tolerance, dependence, and withdrawal exhibited by LC neurons. The proposed studies will further characterize the role of the cAMP pathway in acute and chronic opiate action. One major aim of the studies is to more fully investigate the electrophysiologicaI consequences of the upregulated cAMP system in LC neurons by use of intracellular and whole cell recording techniques. Another major aim is to study the mechanisms by which opiates produce the upregulated cAMP system. Preliminary experiments have revealed regulation of specific signal transduction proteins in the cAMP pathway at the protein and mRNA levels, and this will be pursued further. These studies will include analysis of morphine regulation of the expression of a TH promoter-CAT reporter transgene in a transgenic mouse line. Morphine regulation of mRNAs suggests that morphine action may occur at least in part at the level of gene expression. The proposed studies will, therefore, extend our previous observations of acute and chronic morphine regulation of fos/jun and CREB transcription factors in the LC and further characterize the regulation of transcription factor function in opiate addiction. Opiate regulation of other second messenger and protein phosphorylation systems, observed in preliminary investigations, will also be examined to provide a more complete understanding of the postreceptor adaptations induced in the LC by chronic opiates. Finally, we will use an LC-like transformed cell line as a model in vitro system for molecular characterization of the intracellular pathways which underlie acute and chronic opiate action.